The reverse transcriptase telomerase elongates chromosome 3' ends by additional telomeric repeats, compensating for repeat loss during conventional DNA replication. Restrictions of telomerase function set an upper limit on human somatic tissue renewal, with consequences including immune deficiencies from chronic infection and diseases of bone marrow failure, pulmonary fibrosis, and other disorders from inherited telomerase subunit mutations. Inversely, constitutive over-activation of telomerase is almost universally required for human cancer progression and metastasis. Telomerase is unique among polymerases in its reiterative copying of a hard-wired internal template in the enzyme's integral RNA subunit. Also, unlike all other templated DNA polymerases, telomerase must release product that is single-stranded rather than duplex in order to regenerate the template and allow complementary-strand telomere synthesis. The elaborate catalytic cycle of repeat synthesis required to support telomerase specialization is accomplished by intimate co-folding and functional collaboration of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). In addition, telomerase activity at telomeres and telomerase cellular regulation require numerous other subunits of cellular telomerase holoenzymes that are in general poorly characterized due to scarcity. The long-term objective of Collins lab NIGMS research funding is to determine the molecular and biochemical principles that underlie telomerase enzyme mechanism and cellular action. These goals inform fundamental principles of protein-nucleic acid interaction, ribonucleoprotein biogenesis and function, nucleic acid synthesis, cellular proliferation control, genome stability, and tumorigenesis. The strong Collins lab track record of insights supported by NIGMS funding emerges from parallel studies of telomerase in two enabling model systems: the ciliate Tetrahymena and cultured human cells. Our recent efforts have accomplished innovative telomerase reconstitutions that enable dissection of enzyme mechanism; field-shifting discoveries of cellular telomerase holoenzyme subunits and their functions; and truly landmark determinations of Tetrahymena and human telomerase holoenzyme structures by cryo-EM, made possible by a wide range of accumulated expertise. Future studies will build from this foundation towards the ultimate goal of enabling telomerase manipulation for clinical therapeutics. In the near term, expanded cryo-EM studies of human telomerase throughout its catalytic cycle, combined with other structural and biochemical assays, will identify the determinants of dynamic nucleic acid handling necessary for telomeric repeat synthesis. We will also investigate the mechanism of telomerase activation at telomeres and how telomerase synthesis of single-stranded DNA is coupled to the complementary strand synthesis necessary for telomere stability.